JP6328700B2 - Elemental analysis of organic samples - Google Patents

Description

  The present invention relates to the field of imaging mass spectroscopy, particularly imaging elemental mass spectroscopy. In certain embodiments, the present invention relates to analyzing the distribution of a particular element in a biological sample that has been introduced into the sample as an element tag, or that can occur naturally in the sample.

  It is important to determine the distribution of so-called inorganic elements in a biological sample for a number of reasons. Inorganic elements generally refer to elements other than elements that typically form organic materials such as C, H, N, and O. Usually, the target inorganic element is heavier than oxygen and is typically a metal or metalloid element. As reflected by the fast-growing field of metallomics, the natural distribution of inorganic elements in biological samples reveals important information about biological processes at the gene, protein, and metabolite level. In addition, in an approach called element tagging, some so-called element tags (which may also be referred to as markers) typically contain a specific binding agent (to focus on a specific target or process in a biological system ( For example, antibodies, aptamers, metabolic labels, etc.) can be artificially added to the target in the sample. X-ray fluorescence (XRF), secondary electron spectroscopy (SES), X-ray photoelectron spectroscopy (XPS) to measure the abundance of tag elements such as radiation, light (eg fluorescence or absorption), etc. ), Electron Microprobe Analysis (EMPA), Secondary Ion Mass Spectroscopy (SIMS), Laser Plasma Ionization Mass Spectroscopy (LPI MS), and Inductively Coupled Plasma Mass Spectroscopy (ICP MS) etc. Techniques can be used.

  Compared to mass spectrometry techniques such as SIMS or ICP MS, in the case of fluorescence-based assays, the technique can be bothered by low speed but low sensitivity and limited to one or a few targets per analysis Is done.

  Mass spectroscopic techniques allow for highly parallel multiple measurements of elements using, for example, a multi-collector magnetic sector, time-of-flight, Orbitrap or Fourier transform ion cyclotron resonance analyzer. However, when spatially resolved analysis is required, e.g. tissue imaging, low abundance elements are all of these because the spectrum becomes dominated by strong matrix peaks from the tissue. Poses a challenge to other analyzers. These matrix peaks are derived from polyatomic species constituting most of the tissues whose main elements are not only C, H, N, O but also S, P, alkali metals (Na, K), etc. It can be. Although polyatomic species can in principle be eliminated in an RF-only gas-filled reaction cell (eg, US Pat. No. 5,767,512 and US Pat. No. 7,230,232), such reactions are analytes. Is highly dependent on and may affect the target metal, generally resulting in the loss of these target ions. This is particularly noticeable in imaging applications where the starting amount of analyte is limited from the beginning.

  Since ICP MS by laser ablation (LA / ICPMS) is known to be able to ignore the contribution of polyatomic species, for example, International Patent Application No. 2010/133196, German DE 10354787, International Patent Application No. 0151907, International Patent As shown in application No. 020554057, U.S. Pat. No. 8,274,735, International Patent Application No. 2014/063246, International Patent Application No. 2015128490 and others, it has become one of the preferred methods for elemental imaging of tissue. Acquisition speeds of up to several tens of pixels / second with micrometer (μm) spatial resolution have been demonstrated. Even at such speeds, several hours are still required for acquisition of a single image. However, since most of the transport process takes place at atmospheric pressure and low transport rates, further increases in acquisition speed are limited by the temporary diffusion of the signal due to its diffusion during transport of the sample plume from the surface to the ICP torch. . Atmospheric pressure is essential for ICP operation. In addition to this diffusion, the moving lines can be coated with an aerosol formed by the sample material, resulting in carry-over and contamination of the sample introduction unit. Excess contamination will increase the cost of analysis and maintenance time, as any clinical application requires higher throughput.

  The transfer of the ionization process to a vacuum known in the art of the SIMS or laser plasma ionization approach is a very long scanning process, and thus a long exposure time, due to the relatively low current of the ions of interest created. Bring the need for.

Such low currents of created ions are often caused by relatively low concentrations of natural elements or tags in the cell / tissue matrix, rather than by the low efficiency of ionizing agent or secondary ion creation. This also precludes utilizing other methods of multi-channel elemental imaging such as SES, micro X-ray fluorescence (μXRF). Another problem is the rapid contamination of analyzer components with vacuum chambers and organic matrix materials. For example, if the analysis of only one, typically 5 μm thick tissue piece with an area of 100 mm ² is fully utilized for analysis to meet sensitivity requirements, the instrument can be completely contaminated. . In the case of SIMS, there is a further problem of relatively slow rate of sample removal that slows down the analysis of typical tissue samples that are often at least 3-5 micrometers thick.

In the field of isotope ratio mass spectroscopy (IRMS), particularly when an isotope ratio analyzer is interfaced to a gas chromatography (GC) or liquid chromatography (LC) separation stage, the sample is oxidized to produce CO ₂ , a gas such as NO _x , H ₂ O is generated and analyzed to determine the isotope ratio of elements such as C, N, and / or O. Oxidation is performed according to Z. Muccio and G. P. Can be performed in a combustion furnace as described by Jackson, Isotoporatio mass spectrometry, Analyst 134 (2009) 213-222, or in C.I. Osburn and G.M. St-Jean, Limology and Oceanography: Methods 5 (2007) 296-308 may be involved in the wet chemical oxidation process (eg, in LC-IRMS). For example, “dry” oxidation with UV ozone is also routinely used for removal of contamination on surfaces such as semiconductors, glass and the like.

  The present invention is made against this background.

  The present invention relates to an approach that can be used to process a tissue sample prior to analysis of the tissue sample by any of the methods described above. Carefully controlled oxidation conditions limit the diffusion of heavier elements or tags from their original location, since tissue thickness is typically almost comparable to the required spatial resolution of the analysis However, it can result in gradual removal of the organic matrix. As a result, a much smaller amount of material will be desorbed or removed during sampling while delivering the required analyte and imaging information of interest.

  The present invention provides an improved approach to elemental analysis of organic samples by pre-concentration of the elements to be analyzed (which may be variously referred to herein as elements of interest or analyte elements). These are typically inorganic elements that are present in the sample naturally or through the introduction of tags. This allows various methods for elemental imaging that could be ineffective in previous approaches can be used.

According to one aspect of the invention, a method for imaging one or more analyte elements in an organic sample comprising:
Providing the sample as a layer on a substrate;
Reacting (preferably oxidizing) the sample on the substrate to produce one or more volatile products that leave the sample and enter the gas phase, while one or more analyte elements remain in the sample; Thereby removing most of the sample by weight from the substrate by reaction (preferably oxidation) and enriching or concentrating the remaining sample layer with one or more analyte elements;
Detecting one or more analyte elements in the enriched or enriched sample layer using an imaging element analyzer.

  Preferably, the analyte element is not spatially disturbed by the reaction to greater than the spatial resolution of the imaging analysis. Although some individual analytes can be disturbed by distances greater than this, preferably the analyte elements are spatially disturbed by the reaction on average below the spatial resolution of the imaging analysis.

According to another aspect of the invention, an instrument for imaging one or more analyte elements in an organic sample comprising:
A reaction chamber (preferably an oxidation chamber) for receiving a sample provided as a layer on a substrate,
Electromagnetic radiation for introducing one or more chemical or ionic oxidants into the chamber to oxidize the sample and generate one or more volatile products that leave the sample and enter the gas phase While providing the source and / or inlet, one or more analyte elements remain in the sample and are spatially disturbed by oxidation, thereby removing most of the sample layer by weight from the substrate by oxidation, A reaction chamber in which the remaining sample layer is enriched with one or more analyte elements;
An instrument comprising an imaging element analyzer in a detection chamber for detecting a spatial distribution of the one or more analyte elements in the enriched sample layer is provided.

According to a further aspect of the invention, a dedicated imaging element analyzer for imaging one or more analyte elements in an organic sample, comprising:
A chamber for containing an organic sample containing one or more analyte elements to be imaged, wherein the pressure in the chamber around the sample is in the range of 10 ⁻⁵ to 10 ⁻² mbar; ,
(I) An ion gun for irradiating a sample with a high-intensity beam of primary ions, wherein the primary ions are formed in the ion gun at a pressure of less than 1 mbar, and the ion gun focuses the primary ion beam on the surface of the sample. Irradiating the localized spot on the surface of the sample with (ii) an ion gun for moving the spot to a plurality of positions on the surface of the sample over time according to the localized spot on the top At least one primary irradiation means selected from a laser, preferably a powerful laser, for generating ions and moving the spot over time to a plurality of positions on the surface of the sample;
A gas-filled RF ion guide for receiving generated ions containing analyte elements released from a sample in response to primary irradiation, wherein the mass of the analyte elements is less than the mass range or m / z An RF ion guide that prevents subsequent migration of all ions, and preferably at least some of the generated ions undergo an ionic molecular reaction in the ion guide;
A time-of-flight (TOF) mass analyzer for receiving generated ions or reaction products of generated ions from an RF ion guide, having a repetition rate of at least 5 kHz, preferably 50-100 kHz An analyzer comprising a TOF mass analyzer configured as follows.

According to a further aspect of the invention, an elemental analyzer for mass spectrometry and preferably imaging one or more analyte elements in a sample, comprising:
A chamber for containing a sample containing one or more analyte elements, wherein the pressure in the chamber around the sample is preferably in the range of 10 ⁻⁵ to 10 ⁻² mbar;
A laser for irradiating a localized spot on a surface of a sample and laser plasma ionizing at least one or more analyte elements in the sample, preferably the spots at a plurality of positions on the surface of the sample over time A laser that is intended to move
A reaction cell for receiving ions of one or more analyte elements generated by laser plasma ionization, wherein the composition and emission rate of the ions are changed, preferably reduced, as the ions move through the reaction cell; A reaction cell;
In a mass analyzer, preferably a time-of-flight (TOF) mass analyzer, for receiving ions of one or more analyte elements and / or reaction product ions of one or more analyte elements from a reaction cell There is provided an elemental analyzer comprising a TOF mass analyzer, preferably configured to have a repetition rate of at least 5 kHz.

  Pre-concentration of analyte elements on the substrate, which can be tags, etc., converts the organic matrix or sample material into volatile gases that are removed as waste while leaving the target inorganic elements on the substrate. Implemented by allowing oxidation reactions. Thus, preferably the volatile product is substantially free of analyte elements. Furthermore, the inorganic element species can ultimately be in an oxidized form on the substrate (ie, in the oxidized sample).

  The enriched analyte element in the remaining sample can then be detected using an imaging element analyzer. Detection can be performed at different times and locations (eg, in different chambers) for oxidation. For example, detection is typically performed after oxidation. From detection, an image of the detected element in the sample can then be created. Accordingly, the imaging element analyzer may comprise a data acquisition system that receives input from the detection of one or more elements and creates an image of the one or more elements in the sample. The imaging element analyzer is an apparatus capable of high-speed imaging of a plurality of elements in parallel, preferably an apparatus including a mass analyzer or a polychromator.

  The initial sample is an organic sample, i.e., consists mostly of organic material and contains a small or trace amount of inorganic material containing the analyte element to be detected. It can be any sample containing an organic matrix containing one or more analyte elements that is desired to be detected. The organic matrix constitutes the majority of the mass or weight of the sample. The organic matrix is at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or at least 99.9%, by weight of the sample layer, Or it may constitute at least 99.99% by weight.

  The sample can be a biological sample, ie, biological source. The biological sample can be derived from an organism. The organism can be a plant or an animal or a bacterium. In one preferred application of the invention, the biological sample is tissue and / or individual cells.

  Analyte elements are generally elements other than the elements (C, H, N, and O) that typically form an organic matrix. Typically, the analyte element is heavier than oxygen. Typically, the element is a metal or metalloid element. The element can be a metal that is preferably heavier than a mass of 16. The element can be a heavy metal element. The element may be selected from rare earth elements (lanthanides) or transition metals or late transition metals, or alkali metals, alkaline earth metals, or metalloids. The element can be a radioisotope. For multiple analytes, the elements can be any combination of the above classifications. One or more analyte elements may include two or more different isotopes of the same element.

  One or more analyte elements can occur naturally in a sample, for example, as a trace element in a sample, such as a biological sample. Such elements can also be used as internal standards for improved quantification. One or more analyte elements may be introduced into the sample as element tags using, for example, element tagging methods known in the art. One class of preferred element tags are rare earth elements (particularly lanthanides). The tag can be a radioisotope that can be detected by a radiation analyzer.

  One or more elemental tags can be provided as nanoparticles, nanorods, mass dots, or quantum dots, for example, as described in US 2014/0221241 A1.

  One or more element tags may be provided as purified isotopes of rare earths or other elements, or combinations thereof in predetermined proportions.

  One or more elemental tags can be bound to a binding member that binds to a target in the sample. A binding member can be specific such that it binds to a particular target in the sample. If one or more are present, each element tag (each mass) can be bound to a different binding member that is specific for a particular target in the sample. Thus, there can be multiple different targets. Preferably, each element tag binds to a different specific binding member. One or more elemental tags can be bound to the binding member directly or indirectly (eg, via a linker). The binding member can be selected from staining (eg, fluorescent staining), polypeptide, polynucleotide, antibody, affibody, and aptamer), or SOMAmer ™. The target can be any organic molecule in the sample. In the case of biological samples, the target can be a biomolecule, for example a macromolecule such as those selected from proteins, polysaccharides, lipids, and nucleic acids, and small molecules such as metabolites and natural products. The target can be an antigen. As an example, an element tag can bind to an antibody such that it is bound to an antibody-antigen complex after the antibody has bound to an antigen. The target, or each target, is preferably a biomarker.

  In some embodiments, one or more element tags may be metabolically introduced into a sample, eg, food or support medium. Thus, element tags can form part of a metabolic label.

  Tagging is also described, for example, in US Patent Application Publication No. 2014/106976 and B.C. Bodenmiller et al. , Nature Biotechnology 30 (2012) 858-867, multiple elements may be utilized in a bar code fashion.

  The one or more element tags can include two or more different isotope tags of the same element.

  The sample is prepared as a layer on the substrate. The sample is preferably provided as a thin layer, more preferably at a thickness of (i) 20 μm, or (ii) 10 μm, or (iii) 5 μm, or (iv) 3 μm or less.

  The substrate is typically a slide, for example a flat slide. The substrate or slide can be a metal, glass, or ceramic plate. In some embodiments, the substrate may have a titanium dioxide surface. For example, any of the aforementioned slides or plates may have a titanium dioxide surface. For this purpose, the substrate can be coated with a layer of titanium dioxide, preferably in the form of a titanium dioxide film or immobilized titanium dioxide particles. One of the preferred embodiments of the substrate is a standard microscope glass slide having an indium tin oxide coating known in the art.

  In some embodiments, the sample is fixed or embedded tissue sample, eg, formalin fixed, paraffin embedded (FFPE), preferably cut by a microtome, preferably to a thickness of 3-5 μm. Can include tissue.

  In some embodiments, the sample can include individual cells deposited on the substrate, eg, from a flow cytometer or high content screening device. Cells can be deposited, for example, in a lattice-like pattern (eg, every 50 μm or 30 μm in the X and Y directions). Typical cell sizes in this case can be up to 5 μm or up to 10 μm, or 5-10 μm. An example of a lattice-like sample preparation is shown in International Patent Application Publication No. 2014/063246.

  In some embodiments, the sample may comprise a cell culture on a growth medium, for example a microbial or bacterial culture on a thin layer of growth medium such as agarose. Preferably, the culture is up to 10 μm or up to 20 μm thick. Typically, the growth medium will be thicker than the thickness of this culture. Culture samples can be oxidized as they are (eg, using plasma etching), but oxidation and subsequent sampling may not be as effective for thick growth media. Preferably, such samples should be cut into thin culture layers or close to thin culture layers to improve oxidation and sampling.

  In some embodiments, the sample may be deposited by an automated sampling device (eg, including types of automated sampling devices such as flow focusing, acoustic droplet ejection, guidance, etc.).

  Samples can be collected, for example, by means of an automated sampling device, in or on a lattice-like pattern (eg every 30-50 μm in the X and Y directions), or in or on a microarray or in a multiwell plate. It can be deposited on the substrate as drops.

  In some embodiments, multiple samples (which may be different samples) may be deposited on the substrate at different locations on the substrate, for example, in the lattice-like pattern described above.

  Tagging the sample with one or more analyte elements can be performed before or after, preferably after providing the sample on the substrate.

  In certain embodiments of the invention, the analyte element is not an element tag, but an element that is naturally present in the sample (so-called natural element). Thus, in certain embodiments, the sample remains untreated in the sense that it is not tagged. This type of method is intended to produce a distribution of natural inorganic elements such as metallic elements in a sample, especially heavier natural inorganic elements (for example metal, eg Fe, Zn, Sn etc. in metallomics experiments). Can be used in applications.

  Once sample preparation on the substrate is complete, the sample can be transferred to a processing chamber (eg, an oxidation chamber) for an oxidation step. The sample can optionally be lyophilized prior to this transfer to reduce the amount of water in it. The sample can be transferred to an airtight reaction chamber where it is subjected to one or more preferably strong oxidizing agents. In one embodiment, the oxidation may include heating the sample in an oxygen stream or atmosphere to cause combustion. Many different combustion or oxidation processes can function as long as the analyte element or tag is not spatially disturbed by the process, rather than the desired spatial resolution of the analysis. The finer the spatial resolution of the analysis (eg, 1 micron or 3-5 micron spatial resolution may typically be used), the milder the oxidation process should be. In any case, the formation or boiling of gas bubbles strongly disturbs the element's original spatial distribution and is not allowed. In some embodiments, more intense and rapid oxidation can be used if the required spatial resolution is about tens of microns. Although vapor phase oxidation is preferred, wet chemical oxidation and etching with RF emitting plasma can also be implemented as long as the requirement of low perturbation of the spatial distribution of element tags is still implemented. Several oxidation process combinations can be used to accelerate the enrichment of the remaining sample.

  The hermetically sealed chamber may be a reaction chamber that is separate from the detection chamber in which detection or analysis is performed, and it may be the same chamber as the detection or analysis chamber. Thus, in some embodiments, the oxidation chamber is the same chamber as the detection chamber, i.e., in such cases there is a single oxidation and detection chamber. Preferably, the oxidation is performed in a chamber different from the detection chamber in which the imaging element analyzer is located. Thus, the oxidized sample must typically be transferred from the reaction (ie, oxidation) chamber to the detection / analysis chamber (where the imaging element analyzer is located). Thus, in such cases, once the oxidation process is complete, the substrate (slide) is transferred into the vacuum chamber for elemental imaging by any one of a number of applicable methods (ie, the process is Overall, it is a two step process: conversion / concentration followed by vacuum-based imaging analysis).

  Preferably, the oxidation step includes exposing the sample to one or more oxidizing agents, and optionally the sample is heated during the oxidation step. Thus, the instrument, eg, the reaction chamber, may further include a heater for heating the sample during the oxidation step. The reaction chamber may be a vacuum chamber. Oxidation can be performed in the chamber at elevated pressure (greater than atmospheric pressure), atmospheric pressure, or reduced pressure (ie, less than atmospheric pressure). The reduced pressure state can be between 100 and 1000 mbar or between 1 and 100 mbar. In the latter case, a DC or RF gas discharge may be used to promote oxidation. The one or more oxidants may be (i) electromagnetic radiation, and / or (ii) one or more gas phase chemical oxidants, and / or (iii) ions or electrons, and / or (iv) one or more It can be selected from liquid phase chemical oxidants. Accordingly, one or more chemical oxidant sources may be connected to the inlet, preferably the one or more oxidants are ozone, hydrogen peroxide, and persulfate as an example of a liquid phase oxidant. (Eg, ammonium persulfate, sodium, or potassium). The latter is preferably used with catalysts such as phosphoric acid and silver nitrate. Agents may be placed in the chamber together or continuously at reduced pressure (below atmospheric pressure).

Preferred gas phase oxidants that can be used include ozone and hydrogen peroxide. The sample can be oxidized by the action of electromagnetic radiation, specifically light, in particular light having a wavelength of less than 400 nm (preferably UV light, but in some embodiments X-rays). For the example of ozone oxidation, the UV ozone oxidation chamber may provide atmospheric pressure or atmospheric pressure as known in the art, further providing supplementary activation with wet air or oxygen and UV light at 254 nm and / or 185 nm. Can be used at boost. If oxidation is performed using light, the sample is optionally present on the photocatalytic surface (preferably the titanium dioxide surface) of the substrate subjected to light. Light oxidation is preferably 0.1 milliwatts / cm ^2, greater than or 1.0 milliwatts / cm ^2, greater than or 10 milliwatts / cm ^2, or greater than the surface at an intensity of from 0.1 to 10 milliwatts / cm ² Irradiate.

  Accordingly, the one or more oxidants can be (i) electromagnetic radiation, wherein the oxidation step includes irradiating the sample with light of a wavelength less than 400 nm, and the substrate serves as a photocatalyst that oxidizes the sample. Or (ii) one or more chemical oxidants, wherein the oxidation step comprises exposing the sample to one or more chemical oxidants selected from ozone and hydrogen peroxide.

  Similar effects are achieved by contacting the sample with a low-pressure RF or DC gas discharge so that the surface of the sample is bombarded by charged particles from the plasma and oxidation is assisted by sputtering. This process is typically more suitable for larger lumps or crystals of elements because it is faster and more “wearable” compared to UV ozone treatment.

The oxidation process described is a rapid oxidation of organic matrix atoms, such as any one or more of the following oxidation reactions: C → CO, CO ₂ , N → NO, NO ₂ , H → H ₂ O, etc. It is suitable for causing. Accordingly, the one or more volatile products preferably include one or more oxides of C, H, and / or N. Optionally, S is converted to SO ₂ or other sulfur oxides. The produced volatile oxidation product is preferably pumped out and carries away some, preferably most, of the sample mass. At the same time, analyte elements, such as heavier elements (heavier than O), and in particular metal elements, do not produce volatile products and thus remain on the continuously thinning sample layer, mainly in oxidized form. Thus, the sample becomes or is enriched with one or more analyte elements prior to analysis by the imaging element analyzer. Preferably, the location of the analyte element in the sample on the substrate does not change (at least significantly or substantially) as a result of the reaction. For this reason, the reaction rate can be controlled such that no aeration or boiling occurs and the position of the analyte element does not change. For heavier elements or larger lumps or crystals of such elements, the diffusion is low, but the reaction rate is the length of the diffusion a) 1 × sample thickness D, b) 0.5 * D, c) 2 * Should be chosen low enough to keep below D. The image of the analyte element in the sample so determined thereby represents the distribution of the analyte element in the original sample (before reaction).

  Preferably, the reaction step removes most of the sample layer by weight from the substrate. More preferably, in a preferred order, the reaction is at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or at least 99% by weight of the sample layer, or Remove at least 99.9 wt%, or at least 99.99 wt%. In some embodiments, the reaction step removes between 90 wt% and 99 wt%, or between 90 wt% and 99.9 wt% of the sample layer.

  Preferably, the reaction step removes most of the organic matrix (most preferably greater than 90%, or greater than 95%, or greater than 99% by weight), and typically heavier analyte elements are removed. The oxidation process is continued until it substantially reaches or approaches saturation so that it is sufficiently concentrated for subsequent analysis.

  Preferably, the reaction step comprises controlling the rate of the oxidation process by adjusting the supply of oxidant (including light or ions if used) and / or the temperature of the sample. In some embodiments, the production of at least one of the volatile products and / or their vapor phase concentrates may, for example, stop the oxidation reaction (to minimize occupancy of unwanted products). To determine the time to monitor oxidation integrity), or the rate of oxidation (so that it is not too intense because it can disturb the position of the element or tag if it is too intense) Additional types of samples for process control (eg, using one or more gas sensors) and measuring, for example, the relative content of certain elements or their isotopes in volatile products Can be monitored for diagnosis to obtain information (eg, by-products, contaminants, etc.). Another embodiment of the reaction chamber involves using a sample slide made from a porous inorganic material through which an oxidant (ozone, hydrogen peroxide, persulfate) is forced from a supply below. This approach is more likely to result in aeration and boiling because it relies on the rapid diffusion of these agents through the thin tissue pieces. However, in this case, another porous slide can be positioned just a few micrometers away from the tissue to “capture” the heavier elemental non-volatile oxides carried away by the resulting gas flow. This passage approach allows for a faster oxidation process without any loss of analyte of interest, even if bubbles are formed.

  Thus, in some embodiments, reacting the sample on the substrate comprises passing one or more oxidants through the pores in the substrate from the opposite side of the substrate to the sample to reach the sample. And the presence of a second substrate that is proximate to, but spaced from, and facing the sample, thereby diffusing one or more oxidants through the sample, resulting in a volatile product from the sample. Allowing the non-volatile analyte (heavier) elements and / or their oxides to reach and remain on the surface of the second substrate for detection by the imaging analyzer; Including. If there is a sufficiently small gap, eg, 5-10 micrometers, between the two substrates, the gap will be the heavier elemental space of the analyte in the sample after the element has been transferred to the second substrate. It is such that the global distribution is substantially preserved. At least preferably, the analyte elements that are transferred on average to the second substrate are spatially disturbed by oxidation below the spatial resolution of the imaging analysis to be performed.

  Imaging element analyzers are secondary electron spectrometer (SES), X-ray photoelectron spectrometer (XPS), X-ray fluorescence (XRF) spectrometer, energy dispersive X-ray micro analyzer, radiation analyzer, ion mobility analysis And a mass spectrometer (MS), preferably a mass spectrometer.

  Preferably, detecting one or more elements irradiates a sample concentrated with a primary particle beam, such as ions or photons, focused on a localized spot on the surface of the sample, and then secondary from the spot. Releasing the particles and analyzing the secondary particles to determine the presence and optionally the amount of one or more elements in the spot, the spot being moved over time to multiple locations on the surface of the sample; Thereby, an image of the one or more elements in the sample is obtained, and each position of the spot on the surface of the sample corresponds to a pixel of the image. At least some of the secondary ions are ions that include one or more of the analyte elements. Secondary particles are released from the sample surface (for example, if the secondary particles are already elemental ions or their oxide ions and the analyzer is a mass spectrometer, or the secondary particles are Or if they are photons or electrons emitted from one or more analyte elements that are unique to the above analyte elements) or they can be converted to another form for analysis, eg (for example ( Converted from neutral or ionic polyatomic particles released for mass analysis (in the ICP ion source of the mass analyzer) to monoatomic element ions, or in a reaction or collision cell upstream of the mass analyzer ( It can be analyzed directly as it is converted into a reaction product (via ionic molecules or ion-ion reactions).

  Therefore, the imaging element analyzer creates a primary particle beam, focuses the beam to a localized spot on the surface of the sample, and emits secondary particles from the spot (eg, an ion gun). Or a photon source (e.g., a laser) and a secondary particle analyzer for analyzing secondary particles to determine the presence and optionally amount of one or more elements in the spot. Is configured to move the spot to multiple locations on the surface of the sample over time, thereby obtaining an image of one or more elements in the sample, where each location of the spot on the surface of the sample Corresponds to a pixel. The imaging elemental analyzer may acquire the image at a rate in the range of at least 100 pixels per second, or at least 1000 pixels per second, or 1000 to 10,000 pixels per second.

  Preferably, the primary particles are selected from IR, or visible light, or UV, or X-ray photons, or electrons, and ions. Also preferably, the secondary particles are selected photons (especially X-rays), electrons, and ions.

  Preferably, the energy of the primary particles exceeds 1 keV.

  Preferably, the primary particles are ions, and more preferably the primary particle source comprises an ion gun. Preferably, the primary ion beam is continuous. However, in some embodiments, the primary ion beam is a pulse. Preferably, the primary particles are ions formed at a pressure of less than 1 mbar and the secondary particles are ions for analysis by a mass analyzer. Thus, the imaging elemental analyzer can be an imaging secondary ion mass spectrometer (SIMS) configured to be evacuated to vacuum, where the primary particles are ions formed at the source at a pressure of less than 1 mbar. Secondary particles are ions for analysis by SIMS mass analyzer.

  Sample scanning may be implemented by an ion gun steering plate and / or by moving a stage on which the sample or substrate is positioned. For example, a thinner sample (eg, a tissue section thickness of 3 μm or less) and / or stronger spatial focusing of the primary beam and / or a primary beam current to reduce space charge defocus. By using a higher spatial resolution for intracellular and organelle resolution can be achieved.

  Preferably, the primary ion beam has an intensity of up to 100 nA per 1 μm sized spot (ie 1 μm diameter spot). Preferably, the primary ion beam has an intensity of at least a) 1 pA, or b) 100 pA, or c) 1 nA, or d) 10 nA per 1 μm size spot.

When photons are used as primary particles, preferably the fluence in the ionization pulse exceeds a) 5, b) 10, c) 20, d) 50 J / cm ² , which is known in the art. As such, a high density plasma is formed, allowing a high degree of ionization to be achieved. Preferably, a laser device is used as the photon source. Preferably such a laser is a pulsed laser, preferably the pulse has the fluence described above within each pulse. Any laser wavelength can be used, but is preferably less than the required spatial resolution, and preferably a) an absorption length of 100 nm, b) 200 nm, or c) 500 nm or less (ie, a coefficient of e (Euler The length of the laser intensity) that is attenuated by the number). For example, preferably, the fluence within the irradiation pulse is greater than a) 5, or b) 10 J / cm ² (or other preferred fluence) and has an absorption length of 500 nm or less. Most preferably, a solid state laser such as Nd: YAG is used for ionization, with or without frequency amplification (the latter may be required to obtain a sufficiently short extinction length). .

  The laser emission line can illuminate the sample from the front or back. The latter case provides easier focusing of the laser beam, orthogonality between the laser light and the sample, so that glass that is a circular spot but a high emission threshold is used to avoid damage to the emission line of the slide. Should be used. In the case of front illumination, the laser light is typically incident at an angle (and thus simplifying the more demanding requirements of the extracted ion optics), while optical observation of the focal point is glass Can be done from the back through the slide.

  In embodiments where the imaging element analyzer comprises a mass analyzer for determining the m / z of emitted ions or their reaction products, the mass analyzer is a time-of-flight (TOF) mass analyzer, flight Distance type mass analyzer, quadrupole ion trap mass analyzer, electrostatic trap (EST) mass analyzer (orbital EST, eg Orbitrap), ion cyclotron resonance mass analyzer (FT-ICR), especially imaging current detection and , Or EST or ICR using a magnetic sector mass analyzer and an array, or a combination thereof, more preferably the mass analyzer comprises a TOF mass analyzer. The TOF analyzer has an orthogonal acceleration TOF analyzer (OA-TOF), in particular preferably a latticeless orthogonal acceleration, optionally reflecting single or multiple ions within the analyzer (more preferably a single reflection). A high repetition rate OA-TOF known in the art with one or more ion mirrors. Preferably, the mass analyzer is capable of analyzing a continuous secondary ion beam simultaneously over a wide mass range of ions, such as TOF or Orbitrap or orbital electrostatics. Preferably, for example, there are a plurality of analyte elements to be analyzed (imaged) from a plurality of element tags. More preferably, there are at least 5 or at least 10 analyte elements or element tags that are analyzed by an elemental analyzer. Mass spectrometers, especially TOF mass analyzers, can easily perform such multi-channel analysis on a short time scale. For multi-model analysis, a combination of analyzers is used, for example a TOF mass analyzer for nominal mass elemental analysis, and an electrostatic trap with image current detection such as organic analysis or Orbitrap for high resolution interference Can be done.

Analyte element (tag) secondary ions can be released into an intermediate vacuum pressure of 10 ⁻⁵ to 10 ⁻² mbar rather than a higher vacuum, compared to high vacuum (eg, SIMS) instruments Reduce the requirements for sample drying and transfer time. Thus, the chamber for containing the sample irradiated with primary ions has a pressure in the chamber around the sample in the range of 10 ⁻⁵ to 10 ⁻² mbar.

Preferably, secondary particles for analysis by the mass analyzer are transferred into the RF ion guide after they are released from the surface and delivered from the ion guide to the mass analyzer. Thus, the imaging element analyzer preferably comprises an RF ion guide for receiving secondary ions after they are ejected from the surface and transferring the secondary ions to the mass analyzer. The ion guide used is preferably gas-filled, for example to a pressure of at least 10 ⁻² mbar, so that if oxides of analyte elements or element ions are detected, less than the mass range of those oxides All m / z is excluded. The ion guide can thereby advantageously function as a reaction or collision cell.

In some embodiments, the ion guide may contain a reactive gas to produce a reaction product with secondary particles (eg, via an ion-molecule reaction), where the secondary particles are elemental. Is an ion containing one or more of For example, reactive gases include gases such as NO or O ₂ to fully oxidize most of the analyte elements (eg, natural metals or rare earth elements) to oxides known in the art. (See, for example, G. Koyanagi, D. Bohme. J. Phys. Chem. A, 105 (2001) 8964-8968), and therefore (with their mass peaks moved by 16 amu while others Reducing the interference) (by not moving the peaks of), and increasing the number of channels analyzed in parallel. If a reactive gas is used, all oxides and other molecular interferences can be eliminated. Thus, preferably, at least some of the (secondary) ions that are generated undergo an ionic molecular reaction within the ion guide. The ion guide may have a cooling effect on the ions as they are transmitted through the ion guide. In this method, the energy distribution or emission rate of ions at the output of the ion guide is changed (preferably reduced) compared to the ions at the entrance of the ion guide. Thus, in some embodiments, the composition and / or energy distribution of ions at the output of the ion guide can be altered as compared to the ions at the entrance of the ion guide. Therefore, the ion guide is preferably configured as a reaction cell. It is known in the art that gases such as N ₂ O, NO ₂ , O ₂ , CO ₂ , NO promote the oxidation of many metal ions, especially ions having m / z above 100, This likewise makes it possible to reduce the number of analysis channels per isotope to one. NH ₃ and NO can also be used to remove polyatomic species such as hydrides prior to this oxidation. Thus, the reaction cell can be filled with one or more such gases that serve as one or more reactive gases within the reaction cell.

  In certain embodiments, as heavier elements are concentrated on the slide surface, they may begin to affect the analysis, forming a new kind of matrix that results in reduced ionization yield or tissue distribution related effects. Can do. For example, phospho- and phospholipids can be concentrated around the cell membrane (including the nuclear membrane) and from DNA in the nucleus. Alkali metals (eg, Na, K) can appear at different concentrations on the opposite side, such as a membrane. Thus, in such embodiments, one or more of these elements (eg, P, Na, K) should also preferably be monitored and corrections applied according to their abundance. For example, in some embodiments, if such common element (eg, Na, K, etc.) concentrates do not follow their expected concentration within a known region in the sample, their expected Observed deviations from the concentrations that can be used as a basis for correcting the detected abundance of the analyte (typically heavier) element.

  In addition, further processing can be applied to the sample, such as, for example, applying an additional matrix to improve ionization and compensate for matrix effects. For example, a uniform coating containing one or more elements to aid ionization can be applied to the sample (eg, oxygen is known to increase the yield of metal secondary ions). Also, in the case of laser irradiation and / or ionization, an organic light absorbing layer can be added to the sample.

  Preferably, the mass analyzer is a TOF mass analyzer, in particular OA-TOF, and the repetition rate of the TOF mass analyzer is at least (a) 5 kHz, or (b) 20 kHz, or (c) 50 kHz, or (d) 100 kHz. Or higher. The repetition rate can be, for example, 50-100 kHz. As used herein, repetition rate refers to the rate of pulsed ions into the TOF analyzer for the separation of ions by their mass to charge ratio (m / z). A detected ion signal (intensity versus m / z) is obtained from each pulse of ions into the TOF analyzer. The sum of the detection signals from each pulse of ions into the TOF analyzer at a given spot on the sample surface can be used to create a corresponding pixel of the elemental distribution image. Preferably, the elemental distribution image is acquired at a rate in the range of at least 100 pixels per second, or at least 1000 pixels per second, or 100 to 1000 pixels per second. Preferably, the mass analyzer is configured to detect an element or an oxide of the element.

  Preferably, analysis of each spot using a TOF mass analyzer requires no more than (a) 2, or (b) 5, or (c) 10 pulses of the TOF analyzer. The analysis time for each spot is preferably the time to acquire a sufficient or desired signal to noise ratio, or mass spectrum of sensitivity for use in the pixels of the image.

The present invention can find use in any of the following.
i. Those applied to anatomical conditions, particularly cancer, including a combination of tissue imaging, eg, fixed paraffin-embedded tissue and hematoxylin and eosin (H & E) staining,
ii. High content cell screening,
iii. A microarray-based targeting assay for biomarkers of clinically relevant diseases,
iv. High-throughput medicine, chemistry, and clinical analysis,
v. Bacterial identification and susceptibility testing (in culture on a thin layer of medium)
vi. Cytometry including off-line flow cytometry.

  If the sample is a biological sample, particularly a clinically relevant sample, the method uses an image of one or more elements in the sample to determine the physiological state in the organism from which the biological sample is derived, or It may further comprise diagnosing a disease state.

  The sample preconcentration approach of the present invention increases pixel acquisition speed and hence increases elemental imaging throughput; reduces instrument contamination; applicability with an extended range of elemental imaging techniques; and elemental oxides on the oxidized surface Numerous benefits can be provided, such as increased ionization efficiency due to the presence of ionic species.

1 schematically illustrates one embodiment of an instrument for imaging one or more elements in an organic sample according to the present invention. Fig. 3 schematically illustrates another embodiment of an instrument for imaging one or more elements in an organic sample according to the present invention. Fig. 6 schematically shows a pass-through embodiment with movement of the analyte of interest onto the opposite slide.

  In order to allow a more detailed understanding of the present invention, a number of embodiments will now be described by way of example with reference to the accompanying drawings.

  Referring to FIG. 1, an apparatus for imaging one or more elements in an organic sample is schematically shown. A thin layer of the sample to be analyzed is deposited on the slide (2). The sample can be arranged as a microarray on the slide. The slide is typically a flat glass slide with an ITO coating. Alternatively, it can be a metal plate.

The sample is a biological (organic) sample such as, for example, a tissue sample or a cell line. In general, however, the sample is not limited to a given type. The sample can include any of the following:
A biological or chemical (organic but non-biological) sample; a fixed embedded tissue, for example a fixed or embedded tissue sample, preferably cut to a thickness of 3-5 μm, for example Formalin-fixed, paraffin-embedded (FFPE) tissue—eg, on a slide from a flow cytometer or high content screening device, eg, a lattice-like pattern (eg, for typical cell sizes up to 5-10 μm X And individual cells deposited every 50 μm in the Y direction). Typically, no more than one biological cell occupies each lattice cell or square. The lattice cells may all be different (eg, from different samples or experiments), or at least some, and optionally all, of the lattice cells are 1 to determine mutations within that sample or population. May be from one sample or population-a cell culture on a growth medium, for example a microbial or bacterial culture on a thin layer of a growth medium such as agarose (preferably the culture is up to 10 μm or 20 μm Is the thickness)
A sample, such as a non-cellular sample, deposited on a slide, eg, by any known type of automated sampling device (including by flow focusing, acoustic droplet ejection, guidance, etc.). The sample can be deposited as a lattice-like pattern (eg, every 50 μm in the X and Y directions) or as individual droplets on the microarray.

  For reasons further described below, in some embodiments, the slide is coated with a layer of titanium dioxide, for example, in the form of a film or immobilized particles.

  In some embodiments, the sample on the slide is analyzed as is (ie, untreated, untagged) for heavier natural inorganic elements (for example, Fe, Zn, Sn, etc. in metallomics experiments). The distribution can be determined. In other embodiments, the sample may be tagged with one or more elements that are preferably non-natural to the sample (non-natural elements herein). Rare earth elements are one class of element tags known in the art. One or more tags are typically selective for one or more different respective targets in the sample. The tag may be applied to the sample before or after the sample is deposited on the slide, but is preferably applied after. Tagging specificity can be achieved by the aid of binding members such as antibodies, aptamers, sommers, metabolic labeling, and other known methods. Tags can include polymer chains, nanoparticles (shown in US 8,679,858), quantum dots, and the like. Tagging is also described in US Patent Application Publication No. 2014/106976 and B.C. Bodenmiller et al. , Nature Biotechnology 30 (2012) 858-867, multiple elements may be utilized in a bar code fashion.

The next step in the method of analyzing the sample involves placing the (tagged) sample-containing slide in the reaction or oxidation chamber (10), performing the oxidation reaction to remove most of the organic matrix. Reduces the mass of the sample, thereby leaving the sample containing the analyte element of interest typically as an oxide. One or both of two approaches to oxidation can be used. The first approach uses light of a wavelength less than 400 nm, such as UV light (12), preferably with an intensity of 0.1-10 milliwatts / cm ² or higher and a wavelength of less than 400 nm (eg, the art Irradiating the slide and its sample (from gas discharge sources known in the art). There are preferred embodiments that include a coating of titanium dioxide or other photocatalyst on a slide that is subjected to UV light. Titanium dioxide exhibits very strong photocatalytic properties that result in rapid oxidation of matrix atoms. Alternatively or in addition, a chemical oxidant, preferably ozone or hydrogen peroxide vapor, enters the oxidation chamber (10) via the inlet (14) and is exhausted therefrom through the outlet (16). As is known in the art, the sample may be heated on the slide to aid in the oxidation process.

Under the influence of UV light and / or oxidants, rapid oxidation of matrix atoms occurs, for example, C → CO _2, N → NO, NO ₂ , H → H ₂ O. Volatile products are pumped out by a vacuum pump (not shown) connected to the chamber, thus carrying most of the sample mass away. At the same time, heavier atoms including tag analyte elements or heavier natural elements do not form volatile products and therefore remain on the thin layer of the sample primarily in oxidized form. When the process reaches saturation and most of the organic matrix is removed (preferably greater than 90% or greater than 99%, e.g., 90-99%), heavier atoms are available for subsequent analysis. Fully concentrated. The rate of the oxidation process can be adjusted by supplying oxidant or irradiation light power and / or controlling the temperature of the sample. Undesirably too fast oxidation can cause gas bubbles to carry away heavier atoms of interest. Therefore, the rate of oxidation should be carefully balanced for sample generation in the reaction chamber. Volatile products may provide additional types of information for process control (eg, to determine when to stop oxidation) and / or (eg, by measuring the relative content of elements or their isotopes). Can be used for diagnosis). For example, elements of volatile products can be monitored. For example, if the monitored C to O ratio is 1: 1, this indicates incomplete oxidation, but if the C to O ratio is 1: 2, this indicates complete oxidation to CO ₂ . Indicates. In another example, an isotope ratio, eg, C ¹² / C ¹³ may be used as a process indicator. For example, this ratio is to distinguish between when the process has finished oxidizing the bacterial culture and when the medium has begun to oxidize (which can have different ratios of C ¹² / C ^{13 to} bacteria). Can be used.

  An alternative approach is illustrated in FIG. 3, allowing a faster reaction, which in principle allows further boiling and aeration of the sample. In this case, one or more oxidants (ozone, hydrogen peroxide, persulfate) are supplied from a source (not shown) under a sample (104), eg, a slide (102) that supports a thin tissue sample. (101). In this embodiment, there are two sample slides (102, 106) that are spaced apart but are closely separated, each made from a porous inorganic material (eg, glass, ceramic, ITO, etc.). The One or more oxidants are forced through two sample slides that are closely separated. These agents diffuse rapidly through thin tissue pieces that produce light gas (ie, volatile products) along the way. This mixture continues to flow from the sample slide (102) to the opposite slide (106) and through the latter through a 5-10 micrometer gap. If the sample is sufficiently lyophilized, it can be in direct contact with the opposite slide (106). The size of the opposite slide pore is such that heavier non-volatile elements and their oxides cannot enter the pore and remain on the surface of the opposite slide for subsequent analysis. (Preferably in the range of 1 to 10 nm). Thus, as described herein, imaging analysis can be performed on the opposite slide. With a sufficiently small gap between the slides, the spatial distribution of heavier elements in the sample is substantially maintained. This passage approach allows for a faster oxidation process without any loss of analyte of interest, even if bubbles are formed. Preferably, the opposite slide is transparent in the UV range and assisted by the ozone emission and titanium dioxide reaction (eg when the sample substrate includes a titanium dioxide surface) assisted by UV radiation from a UV source (108). to enable. This can facilitate the oxidation process.

  The reaction can also be facilitated by a focused laser rastered over the surface. In one class of embodiments, the power of the laser can be high enough to produce local heating that accelerates the rate of organic degradation and oxidation.

  It should be understood that multiple sample slides can be processed simultaneously in the oxidation chamber.

Samples are removed from the reaction chamber for subsequent analysis, preferably with multiple acquisition rates at acquisition rates greater than 100 pixels / second or greater than 1000 pixels / second (eg, 100-1000 pixels / second or 2000 pixels / second). These elements are transferred to an apparatus capable of rapid imaging in parallel. In some cases, the speed can be up to 10 ⁵ pixels / second. The pixel size for such acquisition rates is an intracellular resolution of 10 μm or less, or 5 μm or less, or 2 μm or less, or ideally 1 μm or less (eg, 0.5-1.0 μm). The reaction chamber can alternatively be integrated into the imaging device, which means that some means in the laser (eg, an ion or electron gun, or an x-ray gun in an imaging analyzer) is used, for example, to accelerate oxidation. Note that it is preferred when used.

A preferred imaging device in the form of a secondary ion mass spectrometer (SIMS) or LPI (laser plasma ionization) mass spectrometer is shown in FIG. It comprises a high-intensity laser or ion gun (20) as an irradiation means for generating ions from the sample. Referring to FIG. 2, which shares many of the same features as FIG. 1, further details of an ion gun (20) as a primary ion source for illuminating a sample are shown. The ion gun includes an ionization chamber (22), a lens system (24), and optionally a collimator system (26), and focusing optics (28) for focusing the ion beam to a small spot on the sample surface. And a raster electrode plate (30) for scanning the ion spot across the sample. The primary ion beam ionizes element tags or heavier natural elements of interest and releases them from the surface. Laser ablation (LA) or laser plasma ionization (LPI) is used alternatively, preferably with a high fluence of more than 1-10 joules / cm ² to promote dissociation of molecular bonds and release of elemental ions. Also good. Scanning with a continuous primary beam (ion or laser beam) allows imaging to be performed faster than mechanical movement of the sample support is possible. Typically, a combination of rasters with deflection plates over an area of 0.5 × 0.5 or 1 × 1 mm can be combined with a mechanical raster over a larger distance, for example 50 × 100 or 100 × 200 mm.

In general, a continuous high intensity primary ion beam (36) of up to 100 nA at a 1 μm spot is created. For example, a suitable ion source is N.I. S. Smith, Appl. Surf. Science, 255 (2008) 1606-1609), which is an oxygen ion beam created using an RF gas phase ion source. This produces secondary ions (38) from the sample surface containing the element tag at an intermediate vacuum pressure of 10 ⁻⁵ to 10 ⁻² mbar in the chamber containing the sample, compared to a typical SIMS instrument. Reduce any requirements for sample drying and transfer time. In general, ions generated from a sample by SIMS or LPI are generated at an intermediate vacuum pressure of 10 ⁻⁵ to 10 ⁻² mbar in a chamber containing the sample.

The generated ions (38) (or secondary ions if the primary ion is used to irradiate the sample) eliminate all ions (and their oxides) at m / z below the tag mass range. Accelerated through a short gas-filled radio frequency (RF) driven ion guide or collision cell (40) at elevated pressure (typically greater than 10 ^-2 mbar). As ions pass through the guide, the emission rate of the ion beam is also reduced. The RF ion guide typically includes a multipole such as a quadrupole (42) positioned within a gas filled enclosure (44). Unlike U.S. Pat. No. 7,910,882, such boosting in an RF ion guide or collision cell can cause any reactive gas (especially e.g., NO, O _2, etc.) can be used to substantially oxidize most of the metal ions to oxides (eg, G. Koyanagi, D. Bohme. J. Phys. Chem. A). , 105 (2001) 8964-8968, S. Tanner, V. Baranov, D. Bandura, Spectrochimica Acta B, 57 (2002) 1361-1452), by monitoring element or tag oxides Reduce interference and increase the number of channels analyzed in parallel. Thus, in certain embodiments, the ion guide is configured as a reaction cell. Preferably, the RF ion guide has a DC gradient for accelerating and controlling ion transport.

  After passing through the RF ion guide (40), the element tag or natural element generated ions (38) are known in the art but operate at a repetition rate of 50-100 kHz (ie, the sample to be ionized). Mass spectrometry using a fast orthogonal acceleration (OA) TOF mass spectrometer (50) with multiple MS scans per spot (pixel). Preferably, as shown in FIG. 1, the TOF-MS comprises a latticeless orthogonal accelerator (52) described in International Patent Application Publication No. 01/11660, a single stage ion mirror (54), for example, Dynamic range detector comprising an electron multiplier as described in US Pat. No. 6,940,066, US Pat. No. 6,864,479, or US 2013/264474 et al. (56). The generated ions (38) are thereby separated by their m / z and detected as shown. The TOF analyzer allows a wide mass range of ions to be analyzed simultaneously. Preferably, there are a plurality of analyte elements to be analyzed (imaged).

  Scanning the sample surface can be done by steering the (raster) plate of the ion gun, or using a moving mirror in the case of a laser, and / or moving the sample stage (eg, in the x and / or y directions) Implemented by Use a thinner sample (eg, a tissue section thickness of 3 μm or less) and / or stronger spatial focusing of the primary ion beam, and primary beam current to reduce space charge defocus. By means of this, a higher spatial resolution can be achieved for intracellular and organelle resolution.

Due to the use of high primary ion currents or powerful lasers as described above, analyte element ions (or tags) account for a significant proportion of the current, and secondary ion currents from the generated ions can be up to several hundred picoamperes. (PA) can be reached (eg, up to 100, 200, 500, or 1000 picoamps or more). This means that all pixels can be analyzed with only 1 TOF MS pulse, allowing the acquisition rate to approach 10 ⁵ pixels / sec in some applications (eg analysis of iron in brain slices). . Thus, the cost per analysis can be greatly reduced since all slides are analyzed within 1 minute. Images larger than 500 × 500 pixels that are suitable for histological images can be generated. The image can be a 500 × 500 μm region of the sample (eg, having a pixel size of 1 μm). Compared to conventional tagged sample fluorescence detection, the method of the present invention provides readings up to tens to 100 colors or channels at a rate comparable to monochromatic measurements.

  It can be seen from the above description and FIG. 1 that various preferred imaging arrangements can be used to image elements in a reacted or oxidized sample. In one arrangement, a mass spectrometer, preferably, is used because laser plasma ionization is used to enter downstream of the reaction cell (the composition and release rate of the generated ions are preferably changed to smaller mutations). Can generate ions from a sample that enters the TOF mass analyzer. Laser plasma ionization may be scanned across the sample to allow an elemental image of the sample to be obtained. In another arrangement, a beam of primary ions is used to enter the downstream of the reaction cell (the composition and release rate of the generated ions are preferably changed to smaller mutations) so that the mass analyzer A SIMS system that generates secondary ions from a sample that is preferably entered into the TOF mass analyzer may be used. A beam of primary ions may be scanned across the sample to allow an elemental image of the sample to be obtained.

This described method also provides higher absolute sensitivity of the analyte element detection approach due to the increased ion yield due to the presence of oxidized analyte element atoms on the oxidized surface, as is known in the art. Can do. With an ionization efficiency of 0.1 to 1% and low loss transport under vacuum conditions, SIMS or LPI methods, for example, for LA / ICP-MS, during transport of LA / ICP-MS in the latter method High losses (typically resulting in 1 × 10 ⁴ to 1 × 10 ⁵ times the loss) can provide orders of magnitude advantages. On the other hand, the proposed method of sample processing is also perfectly compatible with LA / ICP-MS, which can also reduce sample contamination and carryover.

  The result of the analysis is in an analog or quantitative manner (eg, determining how much of an element or tag is present as a concentration, preferably taking into account matrix effects) or (eg, the presence of an element or tag) Can be presented in a digital or qualitative manner. The results can be assembled by the software into an image, for example an image of the element distribution (and thus the target distribution where the element is tagged to the target in the sample).

The following steps are given as an example of the workflow to which the present invention is applied.
1) preparing a sample, eg, a tissue sample,
2) oxidizing the sample with photocatalytic light and / or a chemical oxidant to remove almost all of the organic matrix;
3) Sample surface oxidized by a continuous current ion gun or focused laser pulse with a spatial resolution of 0.5-10 μm, with an intensity such that the sample is destroyed by constituent elements released as ions from the surface Irradiating, wherein the irradiation spot is scanned over the area of the sample surface desired to be analyzed,
4) Use mass spectrometer detection signals from elemental ions in parallel for each irradiation spot and each irradiation spot (multi-channel detection),
5) Determine the presence or absence of the spatial distribution of elements in the sample based on the mass spectrometry information.

  Variations to such workflows can be made according to the description above, for example, element distribution can be used as a substitute for the corresponding antigen when used as an element tag.

  As an alternative to the primary ion method of ionization of the SIMS imaging analyzer of FIG. 2 described above, there are other ionization methods in vacuum that are useful for mass spectrometry analysis such as laser plasma ionization or laser ablation with post-laser ionization. Can be used. It should be noted that since the laser emission line is alternatively delivered from the back of the slide, its transparency can be utilized to simplify the optical system.

As an alternative to mass spectrometry of ions generated from the sample surface, other, eg non-destructive methods of elemental imaging, eg
-Micro X-ray fluorescence (μXRF) enabling analysis under atmospheric pressure conditions, preferably using multiple element detectors to allow parallel detection-X-ray photoelectron spectroscopy (XPS)
-Electron microprobe analyzer (EMPA), especially integrated with electron microscope-Secondary electron spectroscopy (SES)
-Energy dispersive X-ray microanalysis, preferably using a silicon drift detector, can also be used for imaging of elemental tags.

  Any of the elemental imaging techniques can be combined with other sample imaging modalities (eg, optical imaging). Such optical imaging can be used as an internal standard for improved sample quantification.

  From the above description, the present invention converts an organic matrix, eg C, H, N, O, S, into a volatile species that enters the gas phase and leaves mainly heavier inorganic elements in the sample, especially in oxidized form. It can be understood to include providing a substrate or surface having a thin layer of (biological) organic sample that is subjected to an oxidation process. The resulting sample is subjected to high speed imaging analysis of the remaining heavier elements by mass spectroscopy or other techniques in vacuum to determine the spatial distribution of the elements in the sample.

  In an embodiment, the present invention enables an elemental imaging mass spectrometer capable of delivering cell-inward resolution in combination with higher multiple sample readings with higher throughput and lower cost per analysis. . The present invention is preferably based on vacuum secondary ion or laser plasma mass spectroscopy with time-of-flight mass spectrometry (FIG. 1).

The present invention
-For example anatomical symptoms, in particular tissue imaging applied to cancer,
A microarray-based targeting assay for known clinically relevant disease biomarkers or biomarker panels, and for use in life science research and development,
-High content cell screening,
-High throughput medicine and clinical analysis,
Find applications in many of today's high-growth markets such as bacterial identification and antibiotic susceptibility testing.

  It will be appreciated that variations to the above-described embodiments of the invention may be made while still remaining within the scope of the invention. Each feature disclosed in this specification may be replaced with an alternative feature serving the same, equivalent, or similar purpose, unless stated otherwise. Thus, unless stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

  The use of any and all examples or exemplary languages provided herein ("as examples", "etc.", "eg", "examples", and similar terms) are better It is only intended to illustrate the invention and does not represent a limitation on the scope of the invention unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element that is essential to the practice of the invention.

  As used in this specification, including the claims, the singular forms of the term include the plural and vice versa unless the context indicates otherwise. Is done. For example, unless the context indicates otherwise, singular references herein, including the claims, mean “one or more”.

  Throughout this description and the claims, the terms “comprising”, “including”, “having”, and “containing”, as well as, for example, “comprising”, “comprises”. Variants of the word such as “)” mean “including but not limited to” and are not intended to exclude (not exclude) other components.

  All steps described herein may be performed in any order or simultaneously, unless stated otherwise or context requires otherwise.

  All of the features disclosed in this specification may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. Similarly, features described in non-essential combinations may be used separately (not in combination).

Claims (42)

A method for improving the speed and / or detection sensitivity of imaging one or more analyte elements in an organic sample, comprising:
Providing the sample as a layer on a substrate;
Reacting the sample on the substrate to produce one or more volatile products that leave the sample and enter the gas phase, while the one or more analyte elements are in the sample. Remaining and, on average, spatially disturbed by the reaction below the spatial resolution of the imaging analysis, so that most of the sample layer is removed from the substrate by the reaction by weight, and the remaining sample layer is the one The reacting step enriched with the above analyte elements, and then
Detecting and imaging the one or more analyte elements in the enriched sample layer under vacuum using an imaging element analyzer.   The method of claim 1, wherein the sample is a biological sample selected from tissues, cells, bacterial cultures, and bioorganic solutions.   3. A method according to claim 1 or 2, wherein the one or more elements are naturally present in the sample and / or are introduced into the sample as elemental tags.   The method of claim 3, wherein the one or more element tags comprise one or more rare earth elements, heavy metal elements, and / or radioisotopes.   5. The one or more elemental tags are bound to a binding member selected from a polypeptide, polynucleotide, antibody, affibody, and aptamer, and the binding member binds to a target in the sample. The method described in 1.   6. A method according to any of claims 1-5, wherein the one or more elements are metals or metalloids.   The method according to any one of claims 1 to 6, wherein the layer is a thin film, and in particular has a thickness of (i) 20 µm, (ii) 10 µm, or (ii) 5 µm or less.   The reaction is at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or at least 99.9% by weight of the sample layer. , Or at least 99.99% by weight.   9. A method according to any preceding claim, wherein the reaction comprises exposing the sample to one or more oxidants, and optionally the sample is heated during the oxidation step.   The one or more oxidants are selected from (i) electromagnetic radiation and / or (ii) ionic excited species generated by gas discharge, and / or (iii) one or more chemical oxidants. The method according to claim 9.   The one or more oxidizing agents are (i) electromagnetic radiation, and the oxidation step includes irradiating the sample with light having a wavelength of less than 400 nm, and the substrate serves as a photocatalyst that oxidizes the sample Or (ii) one or more chemical oxidants, wherein the oxidation step exposes the sample to one or more chemical oxidants selected from ozone, persulfate, and hydrogen peroxide. The method of claim 10, comprising steps.   The substrate is a flat slide, in particular a substrate having a surface of metal, coated or uncoated glass, coated or uncoated ceramic slab, or titanium dioxide. The method in any one of.   Reacting the sample on the substrate includes passing one or more oxidants from the opposite side of the substrate to the sample through the pores of the substrate to reach the sample; The method according to claim 1.   There is a second substrate proximate to the sample but spaced from the sample and facing the sample, whereby the one or more oxidants diffuse through the sample and from the sample Producing a volatile product, causing a non-volatile analyte element and / or an oxide of the non-volatile analyte element to reach the surface of the second substrate for detection by the imaging analyzer; The spatial distribution of the analyte elements transferred to the second substrate is averaged below the spatial resolution of the imaging analysis performed by the imaging analyzer by the oxidation. 14. A method according to claim 13, wherein the method is such that it is disturbed.   The method according to claim 1, further comprising creating an image of the detected element in the sample.   The imaging element analyzer is selected from a secondary electron spectrometer, an X-ray photoelectron spectrometer, an X-ray fluorescence spectrometer, an energy dispersive X-ray micro analyzer, an ion mobility analyzer, a radiation analyzer, and a mass spectrometer 16. The method of claim 15, wherein: Detecting the one or more elements comprises:
Irradiating the enriched sample with a beam of primary particles focused on a localized spot on the surface of the enriched sample to emit secondary particles from the spot;
Analyzing the secondary particles to determine the presence and optionally the amount of the one or more elements in the spot;
The spot moves over time to a plurality of positions on the surface of the sample, thereby obtaining an image of the one or more elements in the sample, wherein each position of the spot on the surface of the sample is the image 17. The method according to claim 15 or 16, corresponding to a number of pixels.   18. A method according to any of claims 15 to 17, wherein the image is acquired at a rate in the range of at least 100 pixels per second, or at least 1000 pixels per second, or 1000 to 10,000 pixels per second.   19. A method according to claim 17 or 18, wherein the primary particles are selected from x-ray photons, electrons and ions and the secondary particles are selected from photons, electrons and ions.   20. A method according to any of claims 17 to 19, wherein the energy of the primary particles exceeds 1 keV.   21. A method according to any of claims 17 to 20, wherein the primary particles are ions formed at a pressure of less than 1 mbar and the secondary particles are ions for analysis by a mass analyzer.   22. A method according to any of claims 17 to 21, wherein the beam of primary particles comprises a beam of primary ions and has an intensity of 1 to 100 nA per 1 [mu] m diameter spot. The beam of primary particles is a) 5, or b) 10, or c) 20, or d) greater than 50 J / cm ² , a) 100 nm, or b) 200 nm, or c) 500 nm or less during each pulse. 23. A method according to any of claims 17 to 22, comprising a photon beam from a pulsed laser having a fluence with an absorption length in said sample. The secondary particles are ions, the imaging element analyzer includes a mass analyzer,
The mass analyzer is a time-of-flight (TOF) mass analyzer, a distance-of-flight mass analyzer, a quadrupole ion trap mass analyzer, an electrostatic trap mass analyzer, an ion cyclotron resonance mass analyzer, and a magnetic sector mass. 24. A method according to any of claims 17 to 23, selected from an analyzer, or an array or combination thereof.   25. The secondary particles for analysis by the mass analyzer are transferred into the ion guide after they are released from the surface and delivered from an RF ion guide to the mass analyzer. The method described. The ion guide contains a reactive gas for generating a reaction product with the secondary particles;
The secondary particles are ions comprising one or more of the elements;
26. The method of claim 25, wherein the composition and / or energy distribution of the ions at the output of the ion guide is modified for ions at the entrance of the ion guide.   The mass analyzer is a TOF mass analyzer, and the repetition rate of the TOF mass analyzer is at least (a) 5 kHz or more, or (b) 20 kHz or more, or (c) 50 kHz or more, or (d) 100 kHz or more. Or optionally, the analysis of each spot using the TOF mass analyzer does not require more pulses than (a) 2, or (b) 5, or (c) 10 pulses of the TOF analyzer, 27. A method according to any of claims 24-26. An instrument for improving the speed and / or detection sensitivity of imaging one or more analyte elements in an organic sample,
A reaction chamber for receiving a sample provided as a layer on a substrate;
The reaction chamber oxidizes the sample to produce one or more volatile products that leave the sample and enter the gas phase, while the one or more analyte elements remain in the sample. And, on average, spatially disturbed by the oxidation below the spatial resolution of the imaging analysis, so that, by weight, a majority of the sample layer is removed from the substrate by the oxidation, leaving one or more sample layers remaining. An electromagnetic radiation source and / or an inlet for introducing one or more chemical or ionic oxidants into the chamber to be enriched with
Furthermore, an imaging elemental analyzer in the detection chamber for detecting and imaging an empty during distribution of the one or more elements of the sample layer in a concentrated under vacuum, the instrument.   29. The imaging element analyzer comprises a data acquisition system that receives input from the detection of the one or more elements and creates an image of the one or more elements in a concentrated sample. The instrument described.   The sample is a biological sample selected from tissues, bacteria, and cells, and the one or more elements are naturally present in the sample and / or are introduced into the sample as element tags, 30. A device according to claim 28 or 29.   The oxidation is at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99%, or at least 99.9% by weight of the sample layer. Or at least 99.99 wt%, or between 90 wt% and 99 wt%, or between 90 wt% and 99.9 wt%. 32. The instrument according to any of claims 28 to 31 , wherein the radiation source comprises an ultraviolet light source or an X-ray light source.   33. Any of claims 28-32, wherein one or more sources of chemical oxidants are connected to the inlet, and the one or more oxidants are selected from ozone, persulfate, and hydrogen peroxide. A device according to crab.   The instrument according to any of claims 28 to 33, wherein the imaging element analyzer is selected from a secondary electron spectrometer, an X-ray photoelectron spectrometer, an X-ray fluorescence spectrometer, a radiation analyzer, and a mass spectrometer. .   A primary particle source for generating a beam of primary particles, focusing the beam on a localized spot on the surface of the sample, and emitting secondary particles from the spot; A secondary particle analyzer for analyzing the secondary particles to determine the presence and optionally the amount of the one or more elements in the spot, wherein the primary particle source converts the spot into the sample Moving over time to a plurality of positions on the surface of the sample, thereby obtaining an image of the one or more elements in the sample, wherein each position of the spot on the surface of the sample is the image 35. An instrument according to any of claims 28 to 34, corresponding to a pixel of.   36. Any of the claims 28-35, wherein the imaging element analyzer is capable of acquiring the image at a rate in the range of at least 100 pixels per second, or at least 1000 pixels per second, or 1000-10000 pixels per second. A device according to crab.   37. The instrument of claim 35 or 36, wherein the primary particles are selected from photons, electrons, and ions, the secondary particles are selected from photons, electrons, and ions, and the energy of the primary particles exceeds 1 keV.   The imaging elemental analyzer is an imaging secondary ion mass spectrometer (SIMS) configured to be evacuated to vacuum, wherein the primary particles are ions formed in the source at a pressure of less than 1 mbar; The instrument according to any of claims 35 to 37, wherein the secondary particles are ions for analysis by the mass spectrometer of the SIMS.   The primary particles and secondary particles are ions, the imaging element analyzer includes a mass analyzer, and the mass analyzer includes a time-of-flight (TOF) mass analyzer, a flight distance type mass analyzer, and a quadrupole. 39. The instrument of any of claims 35-38, selected from an ion trap mass analyzer, an electrostatic trap mass analyzer, an ion cyclotron resonance mass analyzer, and a magnetic sector mass analyzer, or an array or combination thereof. .   40. The imaging element analyzer of claim 35-39, comprising an RF ion guide for receiving and transferring the secondary ions to the mass analyzer after the secondary ions are emitted from the surface. The instrument according to any one.   The ion guide is configured to receive a reactive gas that fills the ion guide and generates a reaction product with the secondary ion, wherein the secondary ion is one or more of the elements. 41. The device of claim 40, wherein the device is an ion comprising   The mass analyzer is a TOF mass analyzer, and the repetition rate of the TOF mass analyzer is at least (a) 5 kHz or more, or (b) 20 kHz or more, or (c) 50 kHz or more, or (d) 100 kHz or more. The instrument according to any one of claims 39 to 41.